Although lipids can be extracted directly from frozen cell pellets, recovery and detection of phosphoinositides is compromised if further steps aren't taken prior to extraction.
For instance metabolic enzymes continue to be active in frozen samples if not previously denatured. To avert this issue, we recommend an acid precipitation step with either HCl or trichloracetic acid (TCA). See recommended TCA protocol below: This has the secondary benefit of removing interfering and suppressing ions such as nucleotides, inositol phosphates, sugars, etc. The use of methanol to precipitate cell samples will extract phosphatidyl inositol (PI) and lead to inaccurate ratios of PI to the more highly phosphorylated PIPs.
Sample preparation –TCA precipitation
•Suspend sample in 1-2 mL ice-cold 0.5 M trichloroacetic acid (TCA) solution
•Incubate for 5 min on ice.
•Centrifuge the TCA-treated sample at 20.000 x g for 3 min at +4 ˚C and discard the supernatant.
•Add 1 mL 5% (w/v) TCA + 10 mM EDTA to the pellet, vortex for 30 s, centrifuge as before and repeat.
•To prepare 5% TCA, 10mM EDTA, take 9.3 mL of deionized water, add 0.5 mL of 100% (w/v) TCA and 0.2 mL of 0.5 M EDTA (pH 8.0). To prepare 100% (w/v) TCA, add 45.4 mL of deionized water to 100 g of TCA. The final volume will be 100 mL.
•After completely removing the supernatant, store the pellet at -80 ˚C or on dry ice.
WARNING
Some microfuge tubes are unsuitable for use with these protocols. The best we have found is Eppendorf DNA Lo Bind tubes (https://online-shop.eppendorf.us/US-en/Laboratory-Consumables-44512/Tubes-44515/DNA-LoBind-Tubes-PF-56252.html). These have the strength to undergo the g forces encountered during centrifugation during acid precipitation and are resilient to organic solvent exposure. Chloroform will seep through inferior microfuge tubes. The porosity of inferior tubes may also sequester phospholipids.
For instance metabolic enzymes continue to be active in frozen samples if not previously denatured. To avert this issue, we recommend an acid precipitation step with either HCl or trichloracetic acid (TCA). See recommended TCA protocol below: This has the secondary benefit of removing interfering and suppressing ions such as nucleotides, inositol phosphates, sugars, etc. The use of methanol to precipitate cell samples will extract phosphatidyl inositol (PI) and lead to inaccurate ratios of PI to the more highly phosphorylated PIPs.
Sample preparation –TCA precipitation
•Suspend sample in 1-2 mL ice-cold 0.5 M trichloroacetic acid (TCA) solution
•Incubate for 5 min on ice.
•Centrifuge the TCA-treated sample at 20.000 x g for 3 min at +4 ˚C and discard the supernatant.
•Add 1 mL 5% (w/v) TCA + 10 mM EDTA to the pellet, vortex for 30 s, centrifuge as before and repeat.
•To prepare 5% TCA, 10mM EDTA, take 9.3 mL of deionized water, add 0.5 mL of 100% (w/v) TCA and 0.2 mL of 0.5 M EDTA (pH 8.0). To prepare 100% (w/v) TCA, add 45.4 mL of deionized water to 100 g of TCA. The final volume will be 100 mL.
•After completely removing the supernatant, store the pellet at -80 ˚C or on dry ice.
WARNING
Some microfuge tubes are unsuitable for use with these protocols. The best we have found is Eppendorf DNA Lo Bind tubes (https://online-shop.eppendorf.us/US-en/Laboratory-Consumables-44512/Tubes-44515/DNA-LoBind-Tubes-PF-56252.html). These have the strength to undergo the g forces encountered during centrifugation during acid precipitation and are resilient to organic solvent exposure. Chloroform will seep through inferior microfuge tubes. The porosity of inferior tubes may also sequester phospholipids.